Direct Measurement of Supra-Physiological Levels of Ascorbate in Plasma using a Nanophotometer

High dose intravenously administered vitamin C (ascorbate) is currently being tested in clinical trials as an adjuvant to current standard of care therapies in a variety of cancers. Intravenous infusion is used with a goal to achieve supraphysiological ascorbate concentrations in blood of at least 20 mM, 300 to 500 times normal healthy concentrations (0.04-0.08 mM). These trials need quick and easy access to information on the levels of ascorbate achieved in the blood to make clinical decisions. Previous methods that quantify ascorbate levels in blood require extensive preparation, time, and materials that may not always be present in clinical settings. We developed a new approach to meet this need using direct UV spectroscopy with a nanophotometer. The only preparation required is centrifugation of whole blood to separate the red blood cells from plasma. No more than 3 microliters of plasma are needed; the approach can determine the concentration of ascorbate in the range of 3 – 35 mM; the method is fast and efficient. This approach has already been deployed to gather this information in a clinical trial with lung cancer patients

An assay for the rate of removal of extracellular hydrogen peroxide by cells

Cells have a wide range of capacities to remove extracellular hydrogen peroxide. At higher concentrations of extracellular H2O2 (micromolar) the rate of removal can be approximated by a rate equation that is first-order in the concentration of H2O2 and cell density. Here we present a method to determine the observed rate constant for the removal of extracellular H2O2 on a per cell basis. In the cells examined, when exposed to 20 μM H2O2, these rate constants (kcell) range from 0.46×10−12 s−1 cell−1 L for Mia-PaCa-2 cells (human pancreatic carcinoma) to 10.4×10−12 s−1 cell−1 L for U937 cells (human histiocytic lymphoma). For the relatively small red blood cell kcell=2.9×10−12 s−1 cell−1 L. These rate constants, kcell, can be used to compare the capacity of cells to remove higher levels of extracellular H2O2, as often presented in cell culture experiments. They also provide a means to estimate the rate of removal of extracellular H2O2, rate=−kcell [H2O2] (cells L−1), and the half-life of a bolus of H2O2. This information is essential to optimize experimental design and interpret data from experiments that expose cells to extracellular H2O2

Direct spectrophotometric measurement of supra-physiological levels of ascorbate in plasma

Background: Supra-physiological concentrations of ascorbate, vitamin C, in blood, greater than 1 mM, achieved through intravenous administration (IV), are being tested in clinical trials to treat human disease, e.g. cancer. These trials need information on the high levels of ascorbate achieved in blood upon IV administration of pharmacological ascorbate so appropriate clinical decisions can be made. Methods: Here we demonstrate that in the complex matrix of human blood plasma supra-physiological levels of ascorbate can be quantified by direct UV spectroscopy with use of a microvolume UV–vis spectrophotometer. Results: Direct quantitation of ascorbate in plasma in the range of 2.9 mM, lower limit of detection, up to at least 35 mM can be achieved without any sample processing, other than centrifugation. Conclusions: This approach is rapid, economical, and can be used to quantify supraphysiological blood levels of ascorbate associated with the use of IV administration of pharmacological ascorbate to treat disease

Avoiding horror autotoxicus: The importance of dendritic cells in peripheral T cell tolerance

The immune system generally avoids horror autotoxicus or autoimmunity, an attack against the body's own constituents. This avoidance requires that self-reactive T cells be actively silenced or tolerized. We propose that dendritic cells (DCs) play a critical role in establishing tolerance, especially in the periphery, after functioning T cells have been produced in the thymus. In the steady state, meaning in the absence of acute infection and inflammation, DCs are in an immature state and not fully differentiated to carry out their known roles as inducers of immunity. Nevertheless, immature DCs continuously circulate through tissues and into lymphoid organs, capturing self antigens as well as innocuous environmental proteins. Recent experiments have provided direct evidence that antigen-loaded immature DCs silence T cells either by deleting them or by expanding regulatory T cells. This capacity of DCs to induce peripheral tolerance can work in two opposing ways in the context of infection. In acute infection, a beneficial effect should occur. The immune system would overcome the risk of developing autoimmunity and chronic inflammation if, before infection, tolerance were induced to innocuous environmental proteins as well as self antigens captured from dying infected cells. For chronic or persistent pathogens, a second but dire potential could take place. Continuous presentation of a pathogen by immature DCs, HIV-1 for example, may lead to tolerance and active evasion of protective immunity. The function of DCs in defining immunologic self provides a new focus for the study of autoimmunity and chronic immune-based diseases